SITE-DIRECTED MUTATIONS OF THE FAD-LINKED GLYCEROPHOSPHATE DEHYDROGENASE GENE IMPAIRS THE MITOCHONDRIAL ANCHORING OF THE ENZYME IN TRANSFECTED COS-7 CELLS

COS-7 cells were transfected with the green fluorescent protein (GFP) of Aequorea victoria, human mitochondrial FAD-linked glycerophosphate dehydrogenase (mGDH), a mGDH(wt)-EGFP construct, or two mutant mGDH-pr oteins fused with EGFP. The site of mutation was selected to affect ca tionic amino acids in the peptide signal sequence currently believed t o play a key role in the subcellular distribution of mitochondrial pro teins. All proteins were suitably expressed in the COS-7 cells. Howeve r, an increase in mGDH enzymatic activity above the control value in n ontransfected COS-7 cell homogenates was only observed in cells transf ected with mGDH, indicating that the catalytic activity of mGDH was ma sked in fused proteins, Confocal microscopy documented that, in the ce lls transfected with the mGDI(wt)-EGFP construct, the fusion protein w as located exclusively in mitochondria, this contrasting with the nucl ear labelling of cells expressing the green fluorescent protein alone. The mitochondrial anchoring of the mutated mGDH fused protein was alt ered, this alteration being most obvious in the mGDH(313233)-EGFP muta nt. These findings raise the idea that a conformation change of the mG DH protein, as resulting from either an inherited or acquired alterati on of its amino acid sequence, may affect its subcellular distribution and, hence, modify its immunogenic potential. (C) 1998 Academic Press .